1,8-naphthalimide imidazo{4,5,1-de}acridones with anti-tumor activity

ABSTRACT

The invention provides imidazoacridone compounds of general formula (1) which have cytotoxic and anti-tumor activity. The invention also provides methods of preparing the compounds, and methods of using the compounds for the treatment of cancer or other mammalian diseases characterized by undesirably high levels of cell proliferation. The compounds of the invention are also expected to have utility as research tools.

This application claims the benefit of U.S. provisional application Ser.No. 60/187,991 filed Mar. 7, 2000.

FIELD OF THE INVENTION

The present invention relates to the general fields of pharmaceuticalsand cancer chemotherapy, particularly to the areas of cytotoxicantitumor agents and DNA intercalating agents. The invention alsorelates to medicinal chemistry, and the fields of acridone and1,8-naphthalimide organic chemistry.

BACKGROUND OF THE INVENTION 1. Acridine Intercalators

A number of acridine-based compounds which exhibit antitumor activityhave been reported. Cholody et al. described5-[(amino-alkyl)amino]-imidazo[4,5,1-de]acridin-6-ones as a novel classof antineoplastic agents (Cholody et al., J. Med. Chem. 33:49-52(1990)); 8-substituted5-[(aminoalkyl)amino]-6H-v-triazolo[4,5,1-de]acridin-6-ones as potentialantineoplastic agents (Cholody et al., J. Med. Chem. 33:2852-2856(1990)); and chromophore-modified imidazoacridones and their activityagainst murine leukemias (Cholody et al., J. Med. Chem. 35:378-382(1992)). Capps et al. described2-(aminoalkyl)-5-nitropyrazolo[3,4,5-kl]acridines as a new class ofanticancer agents (Capps et al., J. Med. Chem. 35:4770-4778 (1992)).More recently, an 8-hydroxyimidazo[4,5,1-de]acridin-6-one, C1311, hasentered clinical trials (Burger et al., Br. J. Cancer 74:1369-1374(1996); Idem, Br. J. Cancer 81:367-375 (1999)).

It is believed that DNA is the primary target for these compounds, andthat they bind to DNA by intercalation. There is good evidence thatintercalation of DNA by these drugs disrupts the activity of eukaryotictopoisomerase II (Capranico and Zunino, Eur. J. Cancer 28A:2055-2060(1992); Beck et al., Cancer Chemother. Pharmacol. 34(Supp):S14-S18(1994); Nitiss and Beck, Eur. J. Cancer 32A:958-966 (1996)).

2. Naphthalimide Intercalators

Braña et al. have described naphthalimides with basic side chains whichhave anti-tumor activity (Braña et al., Cancer Chemother. Pharmacol.,4:61-66 (1980); Eur. J. Med. Chem., 16:207-212 (1981); U.S. Pat. No.4,204,063; U.S. Pat. No. 5,183,821). Examples which have reached theclinic include the compounds amonafide (Kornek et al., Eur. J. Cancer,30A:398-400 (1994)) and mitonafide (Rosell et al., Invest. New Drugs,10:171-175 (1992); Llombart et al., ibid., 177-181). Numerous othernaphthalimide derivatives, among them nafidimide and azonafide, havebeen studied as well (Sami et al., J. Med. Chem. 39:4978-4987 (1996) andreferences therein).

3. Acridine and Acridone Bis-intercalators

The strong binding to nucleic acids of bis-intercalators, which containtwo planar aromatic systems joined by suitable linker, has long beenknown (Canellakis et al., Biochim. Biophys. Acta 418:277-283 (1976)).Based upon the anti-tumor activity of the mono-intercalators, which werepresumed to function by DNA intercalation, bis-intercalating compoundshave been intensely studied as potential antitumor agents. It has beengenerally assumed that these compounds function by bis-intercalation ofboth chromophores into DNA.

Chen et al. studied diacridines as potential bifunctional intercalators(Chen et al, J. Med. Chem. 21:868-874 (1978)). Gaugain et al. describedthe synthesis and conformational properties of an ethidium homodimer andan acridine ethidium heterodimer (Gaugain et al., Biochemistry17:5071-5078 (1978)). Sinha et al. described the synthesis and antitumorproperties of bis(quinaldine) derivatives (Sinha et al., J. Med. Chem.20:1528-1531(1977)). Roques et al. described the antileukemic activityof pyridocarbazole dimers (Roques et al., Biochem. Pharmacol.28:1811-1815 (1979)). Wright et al. and Le Pecq et al. describedbis-intercalating diacridines and the relationship of structure to DNABinding (Wright et al., Biochemistry, 19:5825-5836 (1990); Le Pecq etal., Eur. J. Biochem., 180:359-366 (1989). Pelaprat et al. described7H-pyridocarbazole dimers as potential antitumor agents (Pelaprat etal., J. Med. Chem. 23:1336-1343 (1980)). Cholody et al., disclosedbis(imidazoacridone) derivatives active against colon tumor cells(Cholody et al., J. Med. Chem. 38:3043-3052 (1995) and studied themechanism of action (Hernandez et al., Cancer Res. 55:2338-2345 (1995);see also Michejda et al., U.S. Pat. No. 5,508,289 and internationalapplication WO 97/38999, the entire disclosures of which areincorporated herein by reference. The same group of workers alsodisclosed certain bis(triazoloacridone) compounds active against HIVtranscription (Turpin et al., Antimicrob. Agents Chemother. 42:487-494(1998).

4. Naphthalimide Bis-intercalators

Braña et al. have described bis-naphthalimides as a class of antitumoragents (Braña et al., Anti-Cancer Drug Design 8:257-268 (1993)).Kirshenbaum et al. described DMP-840, a bis-naphthalimide with promisingantitumor activity (Kirshenbaum et al. Cancer Res. 54:2199-2206 (1994);and Nitiss et al. discussed the mechanism of action of DMP-840 (Nitisset al., Biochemistry 37:3078-3085 (1998)).

Braña et al., in U.S. Pat. Nos. 4,874,863; 5,616,589; and 5,789,418 (allof which are incorporated herein by reference in their entirety),describe numerous bis(1,8-naphthalimide) compounds which have anti-tumoractivity. Ardecky described similar acenaphthalene-derived bis-imideintercalators (Ardecky et al, U.S. Pat. No. 5,086,059), as did Cherneyand Seitz in U.S. Pat. No. 5,359,070 (both of which are incorporated byreference herein). Cherney et al. have also described benzo- andhetero-fused bis(1,8-naphthalimide) derivatives which have anti-tumoractivity (Cherney et al., Bioorg. Med. Chem. Letters, 7:163-168 (1997);U.S. Pat. No. 5,585,382, incorporated by reference herein). Braña et al.have also disclosed benzo-fused 1,8-naphthalimides derived fromanthracene1,9-dicarboxylic acid (Braña et al., J. Med. Chem., 40:449-45491997)). Sun et al. have described an extensive series ofbis-naphthalimide antitumor agents (Sun et al., WO 92/17453; U.S. Pat.No. 5,206,249; U.S. Pat. No. 5,206,250; U.S. Pat. No. 5,376,664; U.S.Pat. No. 5,488,110; and U.S. Pat. No. 5,641,782, all of which areincorporated herein by reference in their entirety). Weis et al.described bis (1,8-naphthalimide anti-tumor agents having variations inthe linker moiety (Weis et al., U.S. Pat. No. 5,604,095, incorporatedherein by reference).

5. Structure-activity Relationships in DNA Intercalators

Many factors, such as physico-chemical characteristics of the planarchromophores, nature of the linking chain (length, rigidity, andionization state), position of attachment, and other factors, stronglyinfluence the binding with DNA and the biological action of thesecompounds. However, it has been found that although such compoundsexhibit high affinity for DNA, there is little correlation between DNAbinding affinity and cytotoxicity or antitumor activity. For example,some bis-intercalators are cytotoxic, while closely related compoundsare merely cytostatic.

Since structure-activity relationships in the class of bis-intercalatorsin relation to their in vivo antitumor action remain unclear, it has notbeen possible to predict which structures will show such activity, evengiven their binding affinity for DNA. Small structural modifications cansubstantially alter the pharmacological properties of a DNA intercalatorwithout similarly affecting DNA binding. Accordingly, research is stillongoing to find DNA intercalating compounds with high antineoplasticactivity, especially those having selective activity towards specifictumors.

Compounds designed as potential bis-intercalating agents have typicallyconsisted of two identical planar aromatic ring systems (“chromophores”)which are capable of intercalation between the base pairs ofdouble-stranded DNA, joined by a flexible linker of suitable length.Compounds having two identical chromophores are referred to herein as“symmetrical”. Previous workers in the field have generally assumed thatthe mechanism of action of bifunctional intercalators depends uponintercalation of both chromophores into DNA (hence the generic term“bis-intercalator”). Bis-intercalated DNA complexes have in fact beenobserved experimentally (Peek et al., Biochemistry 33:3794-3800 (1994);Shui et al. Curr. Med. Chem. 7:59-71 (2000)).

Accordingly, the design of these agents has most often been based onprevious findings concerning structural requirements forbis-intercalation of symmetrical compounds. Most workers have assumedthat if a given chromophore is discovered to be a very tight-binding DNAintercalator, then the linking of two such chromophores will generate asuperior bifunctional intercalator. Given two identical, linked,tight-binding chromophores, previous workers proceeded to optimize thedistance and geometry between the two by modifying the linker, andsought to obtain additional binding interactions between the linker andthe DNA.

Thus, once a promising chromophore has been identified, symmetricalbis-intercalators are usually prepared, and attention is focusedthereafter on chromophore substituents and on modifications to thelinker moiety in attempts to improve anti-tumor activity. There havebeen a few studies directed at bis(1,8-naphthalimides) which arerendered asymmetric by virtue of differing chromophore substitution,with improvements in solubility and occasional improvements inbiological activity being noted (Cherney et al., U.S. Pat. No.5,359,070, incorporated herein by reference; Idem., Bioorg. Med. Chem.Lett. 7:163-168 (1997); Patten et al., U.S. Pat. No. 5,585,382,incorporated herein by reference). Prior to the present invention,however, there has been very little work directed to substantiallyunsymmetrical bifunctional intercalators.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a novel class of unsymmetricalimidazoacridone-naphthalimide based DNA bifunctional intercalators, andtheir use as antineoplastic agents. The compounds are6H-imidazo[4,5,1-de]acridin-6-ones attached through an amino-containinglinker at the 5-position to the 2-position of a 1,8-naphthalimide. Thesecompounds, having two different chromophores, have been found to beexceptionally potent anti-tumor agents, superior to symmetricalbifunctional intercalators containing either chromophore alone.

The invention is most broadly directed to (a) compounds of structure 1,(b) methods of their preparation, and (c) methods of treating diseasescharacterized by excess cellular proliferation, such as cancer, withthese compounds. The compounds of the invention have general structure:

wherein

A=an alkyl linker, such as for example (CH₂)_(m) or (CHR)_(m);

B=an amino-containing linker, such as for example NR′, NR′(CH₂)_(n)NR″,hexahydropyrimiidine-1,3-diyl, piperazine-1,4-diyl,4-aminopiperidine-1,4N-diyl, or 1,4-diazacycloheptane-1,4-diyl;

D=an alkyl linker, for example (CH₂)_(p) or (CHR)_(p);

Y=any common aromatic substituent, such as for example R, COR, CO₂R,CONRR′, SR, SOR, SO₂R, SO₂CF₃, SO₂NRR′, OR, OCF₃, OCOR, OCONRR′, NO₂,NRR′, CN, Ph, CF₃, NRCOR′, NRCONR′R″, NRC(NR′)NR″R′″, NRCOCF₃, NRSO₂R′,NRSO₂CF₃, or halogen;

R=H, CF₃, lower alkyl, amino-lower alkyl, or hydroxy-lower alkyl;

R′, R″=R, C(O)R, or SO₂R; and

m, n, and p=2-6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general synthetic scheme for preparing certain compoundsof the invention.

FIG. 2 shows the activity of compounds of the invention against thetumor cell lines HCT116, HT29, A549, and MEL SK2 at a concentration of100 nM, in the MMT assay.

FIG. 3 shows the activity of compounds of the invention against thetumor cell line HCT116 at varying concentrations, in the MMT assay.

FIG. 4 shows the activity of the compound of Example 2 against the tumorcell lines HCT116, HT29, A549, and Mel.SK2 at varying concentrations, inthe SRB assay.

FIG. 5 shows the activity of the compound of Example 2 against the tumorcell lines HCT116, HT29, A549, MEL SK2, 6.03, and 10.5 at varyingconcentrations, in the SRB assay.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have observed, in studies on the physico-chemicalinteractions of bis-imidazoacridones with DNA, that they do notuniformly bind by bis-intercalation. In addition, it was noted thattheir biological actions were different from those of ditercalinium,which is a classical bis-intercalator. The results of these studiessuggested that while one chromophore of a “bis-intercalator” did indeedintercalate into DNA, the other was unexpectedly interacting with aDNA-binding protein in vivo. It has now been discovered thatunsymmetrical “bis-intercalators,” referred to herein as “bifunctionalintercalators,” generally have superior cytostatic, cytotoxic, andanti-tumor activity compared with prior art symmetrical compounds.Specifically, it has been discovered that bifunctional intercalatorscomprising one 1,8-naphthalimide moiety and one imidazoacridone moiety,connected by an amino-containing linker, are potent anti-tumor agents.

The terms “alkyl” and “acyl” as used herein are intended to includestraight-chain, branched, and cyclic alkyl and acyl groups. The terms“lower alkyl” and “lower acyl” refer to such groups having from one tosix carbon atoms. For example, n-butyl, t-butyl, and cyclobutyl groupsare all encompassed by the term “lower alkyl” as the term is usedherein.

The invention more specifically provides compounds of structure:

wherein

A=(CH₂)_(m) or (CHR)_(m);

B=NR′, NR′(CH₂)_(n)NR″, hexahydropyrimidine-1,3-diyl,piperazine-1,4-diyl, 4-anminopiperidine-1,4N-diyl, or1,4-diazacycloheptane-1,4-diyl;

D=(CH₂)_(p) or (CHR)_(p);

Y=R, COR, CO₂R, CONRR′, SR, SOR, SO₂R, SO₂CF₃, SO₂NRR′, OR, OCF₃, OCOR,OCONRR′, NO₂, NRR′, CN, Ph, CF₃, NRCOR′, NRCONR′R″, NRC(NR′)NR″R′″,NRCOCF₃, NRSO₂R′, NRSO₂CF₃, or halogen;

R=H, CF₃, lower alkyl, amino-lower alkyl, or hydroxy-lower alkyl;

R′, R″=R, C(O)R, or SO₂R; and m, n, and p=2-6.

In the above formulation, each occurrence of R, R′, and R″ is definedindependently of any other occurrences in the same molecule; Y¹-Y⁴ aredefined independently of one another; and m, n, and p are independent ofone another.

In preferred embodiments, A and D are independently C₂-C₄ alkylene, andare optionally substituted with one or more C₁-C₃ lower alkyl,hydroxy-lower-alkyl, or amino-lower-alkyl groups; B is chosen from thegroup consisting of NR, NNRR′, NRCH₂CH₂NR′, NRCH₂CH₂CH₂NR′, andpiperazine-1,4diyl; Y¹-Y⁴ are each independently chosen from the groupconsisting of R, COR, CO₂R, SO₂R, SO₂CF₃, SO₂NRR′, OCF₃, NO₂, CN, CF₃,and halogen; and R is H or lower alkyl.

In particularly preferred embodiments, m, n, and p are independently2,3, or 4. Another group of particularly preferred compounds are thosewherein Y³ is nitro or amino. Yet another group of particularlypreferred compounds are those wherein B is piperazine-1,4-diyl. The mostpreferred compounds are those in which m and p are independently 2,3, or4 and B is piperazine-1,4-diyl.

With the understanding that the invention is not to be limited by anyparticular theory of its mechanism of operation, it is hypothesized thatthe two chromophores, which are necessary for high biological activityand selectivity, play different roles. One provides a mean for dockingthe drug molecule into specific places on DNA by intercalation, whilethe second directly interacts with proteins in vivo, specifically withone or more mammalian topoisomerase enzymes.

By the methods provided herein, and by obvious modifications thereto,the compounds of this invention may be prepared from the appropriatestarting materials. It is intended that where optical and geometricalisomers are available, the pure isomers and diastereomers, and any andall mixtures thereof, are within the scope of the claims. For example,methods of preparing chiral amino-containing linkers are known in theart or will be obvious to one of ordinary skill in the art. Specificexamples are available in the references cited hereinabove. Theexemplified compounds, and the methods of their preparation, arepresented merely by way of example, and the presentation of selectedexamples is not intended to limit the scope of the invention.

Another object of this invention is to provide methods of making thecompounds of the invention. The compounds may be prepared fromcommercially available starting materials by the general processes shownbelow.

In its most general embodiment, one method of preparing the compounds ofthe invention comprises contacting a compound of structure

with a compound of structure

in a suitable inert solvent such as DMF, DMSO, NMP, or the like. Thetime and temperature required for the reaction will vary, dependinginter alia upon the nature of D, Y³ and Y⁴. Generally, the reactionmixture will be gradually raised in temperature until a suitablereaction rate is obtained. This embodiment will be preferred where Y³and/or Y⁴ are nitro, halogen, or other readily reducible groups.Specific examples of this embodiment are provided below.

In a second, alternative method of the invention, the following generalprocess is provided, which comprises contacting a compound of structure

with a compound of structure

in a suitable inert solvent such as DMP, DMSO, NMP, or the like. Thetime and temperature required for the reaction will again vary,depending inter alia upon the nature of D, Y³ and Y⁴. Generally, thereaction mixture will be gradually raised in temperature until asuitable reaction rate is obtained.

This particular embodiment further comprises the conversion of the nitrogroup into a fused imidazolo ring. This may be accomplished by reductionto an amino group, for example by catalytic hydrogenation or transferhydrogenation, or by chemical reduction with low valent metal species(such as zinc, iron, stannous chloride, or the like), to convert thenitro group into an amino group, followed by condensation with formicacid or a formate ester or orthoester. Preferably, both operations arecarried out simultaneously, by catalytic transfer hydrogenation in thepresence of formate or formic acid as exemplified herein:

Alternatively, in a third method of the invention, the following generalprocess is provided, which comprises contacting a compound of structure

with a compound of structure

wherein Z is F, Cl, or another leaving group, in a suitable inertsolvent such as DMF, DMSO, NMP, or the like. The time and temperaturerequired for the reaction will again vary, depending inter alia upon thenature of Z, Y¹ and Y². Generally, the reaction mixture will begradually raised in temperature until a suitable reaction speed isobtained. This embodiment also requires subsequent conversion of thenitro group into a fused imidazole ring, which may be accomplished asdescribed above.

Compounds where at least one substituent Y is nitro may be converted tocompounds where that substituent is amino, by reductive methodswell-known in the art, such as catalytic hydrogenation and reductionwith low-valent metal species such as Sn(II), Zn(0), Fe(0), and thelike. Likewise, compounds where at least one substituent Y is benzloxyor benzyloxycarbonyloxy can be converted to compounds where thatsubstituent is hydroxy, by hydrogenolysis methods known in the art.Alternatively, Y may be OH throughout the synthetic transformations.

In general, it is anticipated that any of the various 1,8-naphthalimidemoieties, any of the various amino-containing linkers, and any of thevarious imidazoacridone moieties which are known as components of DNAintercalators, can be combined into compounds of the present invention.It is further anticipated that such combinations will for the most partbe capable of intercalating into DNA, and that the majority of suchintercalating combinations will be capable of inhibiting topoisomeraseactivity. Accordingly, all such compounds are conceived of as beingwithin the scope of the invention.

The starting heterocyclic systems required for these preparative methodsare either commercially available, or readily prepared by knownsynthetic methods. For example, 1,8-naphthalic anhydride is commerciallyavailable, and there are published methods for preparation of varioussubstituted derivatives. Similarly, numerous published methods forpreparation of imidazo[4,5,1-de]acridones are available. Representativedetailed procedures of the methods of synthesis are provided in theexamples below.

Another object of this invention is to provide a method of treating amammal suffering from cancer, or another disease characterized byundesirable cell proliferation, with the compounds of the invention. Themethod of the invention comprises administering to an individual mammala therapeutically effective amount of at least one compound of formula

or a prodrug or pharmaceutically acceptable salt thereof, which issufficient to inhibit the undesired cell proliferation or tumor growth.

The dose of the compound used in the treatment of such disease will varyin the usual way with the weight and metabolic health of the patient,the severity of any side-effects, and the relative efficacy of thecompound employed when used against the type of tumor involved. Thepreferred initial dose for the general patient population will bedetermined by routine dose-ranging studies, as are conducted for exampleduring clinical trials. Therapeutically effective doses for individualpatients may be determined by titrating the amount of drug given to theindividual to arrive at the desired therapeutic effect without incurringan unacceptable level of side effects, as is currently and routinelydone with other forms of chemotherapy.

For example, a preferred initial dose may be estimated to be betweenabout 10 and 2000 mg/day for an adult human, more preferably between 100and 1000 mg/day. The initial dose may be varied so as to obtain theoptimum therapeutic effect in the patient, and may be provided as adaily dose, in a divided dose regimen, or by continuous infusion.

Administration of the compounds of this invention may be by any methodused for administering therapeutics, such as for example oral,parenteral, intravenous, intramuscular, subcutaneous, or rectaladministration.

This invention also provides pharmaceutical compositions useful forproviding anti-tumor activity, which comprise at least one compound ofthe invention. In addition to comprising at least one of the compoundsdescribed herein, or a pharmaceutically acceptable addition salt orpro-drug thereof, the pharmaceutical composition may also compriseadditives such as preservatives, flavorants, excipients, fillers,wetting agents, binders, disintegrants, buffers, and/or carriers.Suitable additives may be for example magnesium and calcium carbonates,carboxymethylcellulose, starches, sugars, gums, magnesium or calciumstearate, coloring or flavoring agents, and the like. There exists awide variety of pharmaceutically acceptable additives for pharmaceuticaldosage forms, and selection of appropriate additives is a routine matterfor those skilled in art of pharmaceutical formulation.

The compositions may be in the form of tablets, capsules, powders,granules, lozenges, suppositories, reconstitutable powders, or liquidpreparations such as oral or sterile parenteral solutions orsuspensions.

In order to obtain consistency of administration it is preferred that acomposition of the invention is in the form of a unit dose. Unit doseforms for oral administration may be tablets, capsules, and the like,and may contain conventional excipients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; and carriers or fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine. Additivesmay include disintegrants, for example starch, polyvinylpyrrolidone,sodium starch glycolate or microcrystalline cellulose; preservatives,and pharmaceutically acceptable wetting agents such as sodium laurylsulfate.

In addition to unit dose forms, multi-dosage forms are also contemplatedto be within the scope of the invention. Delayed-release compositions,for example those prepared by employing slow-release coatings,micro-encapsulation, and/or slowly-dissolving polymer carriers, willalso be apparent to those skilled in the art, and are contemplated to bewithin the scope of the invention. For example, the compounds of thisinvention may be incorporated into biodegradable polymers allowing forsustained release, the resulting compositions preferably being implantedwhere delivery is desired, for example, at the site of a tumor.Biodegradable polymers suitable for this embodiment are well-known inthe art, see for example Brem et al., J. Neurosurg. 74:441-446 (1991).The compounds of this invention may be also be incorporated into othersustained-release formulations, such as those employing coatedparticles. See for example U.S. Pat. No. 5,968,551 (which isincorporated by reference herein) and references therein.

The solid oral compositions may be prepared by conventional methods ofblending, filling, tabletting or the like. Repeated blending operationsmay be used to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are conventionalin the art. The tablets may be coated according to methods well known innormal pharmaceutical practice, for example with an enteric coating.

Oral liquid preparations may be in the form of, for example, emulsions,syrups, or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminumstearate gel, and hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles(which may include edible oils), for example almond oil or fractionatedcoconut oil, oily esters such as esters of glycerin, propylene glycol,or ethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid; and if desired conventional flavoringor coloring agents.

For parenteral administration, which will be a preferred route ofadministration in the hospital or cancer clinic environment, fluid unitdosage forms are prepared utilizing the compound and a sterile vehicle.Depending on the concentration used, the compound can be eithersuspended or dissolved in the vehicle. In preparing solutions thecompound can be dissolved in water or saline for injection and filtersterilized before filling into a suitable vial or ampoule and sealing.Advantageously, additives such as a local anaesthetic, a preservativeand buffering agents can be dissolved in the vehicle. Suitable bufferingagents are, for example, phosphate and citrate salts. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum.

Parenteral suspensions are prepared in substantially the same manner,except that the compound is suspended in the vehicle instead or beingdissolved, and sterilization accordingly cannot readily be accomplishedby filtration. The compound can be sterilized by filtration of analcohol solution, or by other conventional means, for example byexposure to radiation before or after being suspended in the sterilevehicle. Advantageously, a surfactant or wetting agent is included inthe composition to facilitate uniform distribution of the compound andstability of the suspension.

All references cited in this disclosure are incorporated by referenceherein, in their entirety.

EXAMPLES 1. Synthesis of Compounds

Commercial reagents were purchased from Aldrich Chemical Company(Milwaukee, Wis.). All commercial solvents and reagents were usedwithout further purification. Column chromatography was performed on70-230 mesh silica gel. Melting points were determined on anElectrothermal capillary melting point apparatus and are uncorrected. ¹HNMR spectra were recorded on a Varian VXR-S spectrometer operating at500 MHz, using TMS as an internal standard. Elemental analyses werewithin ±0.4% of theoretical values for C, H, and N. Starting materialsmay be prepared according to, or in analogy to, various publishedprocedures. See for example Capps et al., EP application 145226 (1985);Tarasov et al., Photochem. Photobiol. 70:568-578 (1999); Cholody et al.,J. Med. Chem. 38:3043-3052 (1995); Idem., EP Application 0502668 (1992);Michejda et al., U.S. Pat. No. 5,508,289; Michejda et al., PCTapplication WO97/38999 (1997); and other documents referenced herein.

6-Chloro-2-[(4-Fluorophenyl)Amino]-3-nitrobenzoic Acid

A mixture of 2,6-dichloro-3-nitrobenzoic acid (18.88 g, 0.08 mol),4-fluoroaniline (26.8 g, 0.18 mol) and EtOH (50 ml) was refluxed for 30hours. The solvent was evaporated, benzene (100 ml) and 2N aqueous NaOH(150 ml) were added to the residue, and the mixture was vigorouslystirred for 1 hour. Undissolved material was separated by filtration,the aqueous layer was isolated, and traces of benzene were removed bypartial evaporation. The solution was then made acidic by addition ofconcentrated hydrochloric acid. The resulting yellow precipitate wascollected by filtration and washed with water (100 ml). After drying,the crude material was crystallized from toluene to give 15.36 g (62%)of 7: mp 216-220° C. Anal. (C₁₃H₇N₂O₄ClF) C, H, N.

By this method, beginning with the appropriate anilines, the followingcompounds can also be prepared:

6-chloro-2-(4-methylphenyl)amino-3-nitro-benzoic acid,

6-chloro-2-(4-methoxyphenyl)amino-3-nitrobenzoic acid,

6-chloro-2-(4-benzyloxyphenyl)amino-3-nitrobenzoic acid,

6-chloro-2-(3-methylphenyl)amino-3-nitrobenzoic acid,

6-chloro-2-(3-methoxyphenyl)amino-3-nitrobenzoic acid,

6-chloro-2-(4-cyanophenyl)amino-3-nitrobenzoic acid,

6-chloro-2-(3-cyanophenyl)amino-3-nitrobenzoic acid,

6-chloro-2-[4-(methoxycarbonyloxy)phenyl]amino-3-nitrobenzoic acid,

6-chloro-2-[4-(methanesulfonyl)phenyl]amino-3-nitrobenzoic acid,

6-chloro-2-[4-(trifluoromethoxy)phenyl]amino-3-nitrobenzoic acid,

and others.

1-Chloro-7-Fluoro4-nitro-10H-acridin-9-one (1b)

A mixture of 6-chloro-2-[(4-fluorophenyl)amino]-3-nitrobenzoic acid(12.39 g, 0.04 mol), chloroform (100 ml), and POCl₃ (60 ml, 0.64 mol)was stirred at reflux for 8 h. Solvents were removed under reducedpressure. To the residue was added 200 ml of a mixture of 1,4-dioxaneand water (8:1), and the mixture was acidified with concentratedhydrochloric acid and stirred at reflux for 2 h. Water was added (200ml) and the precipitate was collected by filtration and crystallizedfrom N,N-dimethylformamide—water to give 10.2 g (87%) of 1b as orangeneedles: mp 287-291° C. Anal. (C₁₃H₆N₂O₃ClF) C, H, N.

By this method, but starting with the appropriate6-chloro-2-arylamino-3-nitrobenzoic acids, and separating isomers byrecrystallization and/or column chromatography where needed, thefollowing can be prepared:

1-chloro-7-methyl-4-nitro-10H-acridin-9-one,

1-chloro-7-methoxy-4-nitro-10H-acridin-9-one,

1-chloro-7-benzyloxy-4-nitro-10H-acridin-9-one,

1-chloro-6-methyl-4-nitro-10H-acridin-9-one,

1-chloro-6-methoxy-4-nitro-10H-acridin-9-one,

1-chloro-7-cyano-4-nitro-10H-acridin-9-one,

1-chloro-6-cyano-4-nitro-10H-acridin-9-one,

1-chloro-7-methoxycarbonyloxy-4-nitro-10H-acridin-9-one,

1-chloro-7-methanesulfonyl-4-nitro-10H-acridin-9-one,

1-chloro-7-trifluoromethoxy-4-nitro-10H-acridin-9-one,

and others.

1-{3-[4-(3-Aminopropyl)Piperazin-1-yl]propyl}Amino-7-fluoro-4-nitro-10H-acridin-9-one.(Compound 2d)

A mixture of 1-chloro-7-fluoro-4-nitro-10H-acridin-9-one (2.93 g, 0.01mol), N,N-dimethylformamide (50 ml), and1,4-bis(3-aminopropyl)piperazine (10.0 g, 0.05 mol) was stirred at roomtemperature for 20 hours. Water (250 ml) was added, and the reactionmixture was stirred thoroughly and left overnight in a refrigerator. Thefine precipitate was collected by centrifugation, transferred into 2%hydrochloric acid (300 ml), and stirred for 2 hours. Undissolvedmaterial was separated by centrifugation. The solution was made alkalineand the product was extracted with chloroform (3×100 ml). The chloroformextract was dried and evaporated. Crude product was crystallized fromtoluene-hexane to give 2.78 g (61%) of 2d as a yellow solid: mp 130-134°C. Anal. (C₂₃H₂₉N₆O₃F) C, H, N.

Beginning with N,N-bis(3 -aminopropyl)methylamine,1-{3-[methyl(3-aminopropyl)amino]propyl}amino-7-fluoro-4-nitro-10H-acridin-9-one(compound 2c) is prepared by the above method. By the same method, butbeginning with 1,4-bis(2-aminoethyl)piperazine, it is possible toprepare1-{2-[4-(2-aminoethyl)piperazin-1-yl]ethyl}amino-7-fluoro-4-nitro-10H-acridin-9-one.

5-{3-[4-(3-Aminopropyl)Piperazin-1-yl]propyl}Amino-8-fluoro-6H-imidazo[4,5,1-de]Acridin-6-one (Compound3d)

A solution of 2d (1.37 g, 0.003 mol) in 88% formic acid (30 ml) washydrogenated over Raney nickel (0.8 g) under H₂ at 1 atm overnight. Thereaction mixture was filtered to remove the catalyst. To the filtrateconcentrated hydrochloric acid (3 ml) was added and the mixture wasstirred at reflux for 8 hours. The reaction mixture was concentrated toabout 10 ml and the product was precipitated as a salt by addition ofacetone (50 ml). After drying the precipitate was dissolved in water (10ml) and chromatographed on a preparative HPLC reverse phase column witha gradient of 0.5% TFA in water:methanol (95:5 to 40:60). The majorfraction was collected and made alkaline, and the product was extractedwith chloroform (3×100 ml). After evaporation of solvent the product wascrystallized from benzene-hexane to give 0.741 g (59%) of 3d as a yellowcrystalline powder: mp 126-130° C.; ¹H NMR (CDCl₃) 8.99 (t, 1H), 8.50(s, 1H), 8.20 (m, 1H), 7.97 (d, 1H), 7.92 (m, 1H), 7.52 (m, 1H, C9-H),6.81 (d, J=9.0 Hz, 1H, C4-H), 3.50 (m, 2H), 2.75 (t, 2H), 2.52 (m, 10H),2.42 (t, 2H), 1.96 (m, 2H), 1.65 (m, 2H).Anal. (C₂₄H₂₉N₆OF) C, H, N.

General Procedure for the Preparation of Examples 1-4

A mixture of 3-nitro-1,8-naphthalenedicarboxylic anhydride (0.001 mol)and the, appropriate amine 3 (0.001 mol) is stirred at 80° C. indimethylformamide (8 ml) until the reaction is complete by TLC. Theprecipitated solid is filtered, washed, and dried, and may be purifiedby column chromatography or by crystallization to yield the followingcompounds. By similar methods, various other substituted1,8-naphthalene-dicarboxylic anhydrides may be converted into analogouscompounds.

Example 15-{3-{N-[3-(1,3-dioxo-5-nitro-1H-benz[de]isoguinolin-2-yl)propyl]-methylamino}propyl}amino}-6H-imidazo[4.5.1-de]Acridin-6-one

Purified by silica gel column chromatography using chloroform-methanol(8:1) mixture as eluent: yield 74%, mp 218-221 ° C.; ¹H NMR (CDCl₃) 9.23(d, 1H), 9.04 (d, 1H), 8.97 (t, 1H), 8.71 (m, 1H), 8.50 (s, 1H), 8.34(m, 1H), 7.94 (d, 1H), 7.88 (m, 2H), 7.77 (m, 1H), 7.49 (m, 1H), 6.77(d, 1H), 4.27 (m, 2H), 3.50 (qt, 2H), 2.56 (t, 4H), 2.30 (s, 3H), 1.95(m, 4H). Anal. (C₃₃H₂₈N₆O₅) C, H, N.

Example 25-{3-{4-[3-(1,3-Dioxo-5-nitro-1H-benz[de]isoguinolin-2-yl)propyl]-piperazin-1-yl}propyl}amino}-6H-imidazo[4,5,1-de]acridin-6-one

Orange crystals after crystallization from dimethylformamide-water:yield 82%, mp 227-230° C.; ¹H NMR (CDCl₃) 9.31 (d, 1H), 9.13 (d, 1H),8.97 (t, 1H), 8.76 (m, 1H), 8.56 (m, 1H), 8.55 (s, 1H), 8.42 (m, 1H),7.94 (m, 3H), 7.89 (m, 1H), 7.54 (m, 1H), 6.78 (d, 1H), 4.29 (m, 2H),3.45 (qt, 2H), 2.53 (t, 2H), 2.48 (br m, 4H), 2.39 (t, 2H), 2.34 (br m,4H), 1.96 (m, 2H), 1.88 (m, 2H). Anal. (C₃₆H₃₃N₇O₅) C, H, N.

Example 35-{3-{N-[3-(1,3-dioxo-5-nitro-1H-benz[de]isoguinolin-2-yl)propyl]-methylamino}propyl}amino}-8-fluoro-6H-imidazo[4,5,1-de]acridin-6-one

A mixture of 3-nitro-1,8-naphthalenedicarboxylic anhydride (0.001 mol)and the amine 3c (0.001 mol) is stirred at 80° C. in dimethylformarnide(8 ml) until the reaction is complete by TLC. The precipitated solid isfiltered, washed, dried and purified by column chromatography.

Example 45-{3-{4-[3-(1,3-Dioxo-5-nitro-1H-benzrde]isoguinolin-2-yl)propyl]-piperazin-1-yl}propyl}amino}-8-fluoro-6H-imidazo[4,5,1-de]acridin-6-one

Crystallized twice from dimethylformamide-water: yield 69%, mp 238-240 °C.; ¹H NMR (CDCl₃) 9.31 (d, 1H), 9.13 (d, 1H), 8.96 (t, 1H), 8.78 (m,1H), 8.51 (s, 1H), 8.43 (m, 1H), 8.22 (m, 1H), 7.97 (d, 1H), 7.93 (m,2H), 7.53 (m, 1H), 6.79 (d, 1H), 4.29 (m, 2H), 3.47 (qt, 2H), 2.53 (t,2H), 2.48 (br m, 4H), 2.39 (t, 2H), 2.34 (br m, 4H), 1.96 (m, 2H), 1.88(m, 2H). Anal. (C₃₆H₃₂N₇O₅F) C, H, N.

Example 55-{3-{N-[3-(5-amino-1,3-dioxo-1H-benz[de]isoguinolin-2-yl)propyl]-methylamino}propyl}amino}-6H-imidazo[4,5,1-de]acridin-6-one

To a stirred solution of the compound of Example 1 (0.001 mol) inglacial acetic acid (25 ml) is added stannous chloride (1.52 g, 0.008mol) dissolved in concentrated hydrochloric acid (5 ml). The mixture isstirred at 60 ° C. for 2 h. After cooling, acetone (50 ml) is added andthe mixture is stirred vigorously. The precipitate is collected byfiltration, washed with acetone, and suspended in water (250 ml). Thesuspension is made basic (pH˜12) with sodium hydroxide and the productis extracted with chloroform (5×50 ml). The crude product ischromatographed on silica gel with a chloroform-methanol (10:1) mixturecontaining 0.5% isopropylamine to provide the title compound.

Example 65-{3-{4-[3-(5-amino-1,3-dioxo-1H-benz[de]isoguinolin-2-yl)propyl]-piperazin-1-yl}propyl}amnino}-6H-imidazo[4,5,1-de]acridin-6-one

To a stirred solution of the compound of Example 2 (0.644 g, 0.001 mol)in glacial acetic acid (25 ml), stannous chloride (1.52 g, 0.008 mol)dissolved in concentrated hydrochloric acid (5 ml) was added. Themixture was stirred at 60 ° C. for 2 h. After cooling, acetone (50 ml)was added and the mixture stirred vigorously. The precipitate wascollected by filtration, washed with acetone, and suspended in water(250 ml). The suspension was made basic (pH12) with sodium hydroxide andthe product was extracted with chloroform (5×50 ml). The crude productwas chromatographed on a silica gel column with chloroform-methanol(10:1) mixture containing 0.5% isopropylamine. The main fraction, afterevaporation of solvents, gave 0.550 g (89%) of the title compound as ayellow solid: mp 219-222° C.; ¹H NMR (CDCl₃) 8.99 (m, 1H), 8.57 (m, 1H),8.55 (s, 1H), 8.31 (m, 1H), 8.02 (d, 1H), 7.97 (m, 1H), 7.92 (m, 2H),7.80 (m, 1H), 7.60 (m, 1H), 7.54 (d, 1H), 7.29 (m, 1H), 6.79 (d, 1H),4.24 (m, 2H), 4.17 (s, 2H), 3.46 (qt, 2H), 2.51 (t, 2H), 2.48 (br m,8H), 2.42 (t, 2H), 2.34 (br m, 4H), 1.93 (m, 2H), 1.87 (m, 2H).

Example 75-{3-{N-[3-(5-Amino-1,3-dioxo-1H-benz[de]isoguinolin-2-yl)propyl]-methylamino}propyl}amino}-8-fluoro-6H-imidazo[4,5,1-de]acridin-6-one

To a stirred solution of the compound of Example 3 (0.001 mol) inglacial acetic acid (25 ml) is added stannous chloride (1.52 g, 0.008mol) dissolved in concentrated hydrochloric acid (5 ml). The mixture isstirred at 60 ° C. for 2 h. After cooling, acetone (50 ml) is added andthe mixture is stirred vigorously. The precipitate is collected byfiltration, washed with acetone, and suspended in water (250 ml). Thesuspension is made basic (pH˜12) with sodium hydroxide and the productis extracted with chloroform (5×50 ml). The crude product ischromatographed on silica gel with a chloroform-methanol (10:1) mixturecontaining 0.5% isopropylamine to provide the title compound.

Example 85-{13-{4-[3-(5-Amino-1,3-dioxo-1H-benz[de]isoguinolin-2-yl)propyl]-piperazin-1-yl}propyl}amino}-8-fluoro-6H-imidazo[4,5,5,1-de]acridin-6-one

This compound was obtained by reduction of the compound of Example 4, inan analogous manner to the method of Example 6. Yield: 63% after columnchromatography, mp 240-243° C.; ¹H NMR (CDCl₃) 8.78 (m, 1H), 8.51 (s,1H), 8.31 (m, 1H), 8.22 (m, 1H), 8.02 (d, 1H), 7.97 (m, 1H), 7.92 (m,2H), 7.60 (m, 1H), 7.29 (d, 1H), 6.79 (d, 1H), 4.24 (m, 2H), 4.16 (s,2H), 3.46 (qt, 2H), 2.51 (t, 2H), 2.48 (br m, 8H), 2.43 (t, 2H), 2.34(br m, 4H), 1.94 (m, 2H), 1.88 (m, 2H). Anal. (C₃₆H₃₄N₇O₃F) C, H, N.

2. Biological Studies

Cancer Cell Lines. Human colon carcinoma HCT116 and HT29, human melanomaMELSK2, human lung cancer A549, human leukemia HL60 and human breastcancer MCF₇ cells were purchased from the American Type CultureCollection (Rockville, Md.). Human pancreatic tumor cell lines 6.03 and10.05 were a gift from Dr. Elizabeth Jaffe, Johns Hopkins University.

In Vitro Cytotoxicity Studies. Cells were seeded in quadruplicate foreach studied concentration in 96-well plates (100 μL of mediumcontaining 2000-2500 cells per well) and were allowed to grow for 24 h(day 0). Stock solutions (2.5 mM) of test compounds were preparedfreshly by dissolving their free base forms in distilled water-DMSO(50:50) mixture containing two equivalents of methanesulfonic acid andthen diluted in distilled water to the concentration of 500 μM. Thesesolutions were used to prepare 2 μM working solution and its 10-foldserial dilutions in appropriate media. 100 μL of drug containing mediumor vehicle (control) was added to each well on day 1. The cytotoxicitywas determined using two different methods: the MTT-based, CellTiter96™Non-Radioactive Cell Proliferation Assay (Promega Inc., Madison, Wis.)according to instructions provided by the manufacturer, and/or thesulforhodamine B (SRB) method (Skehan et al., J. Natl. Cancer Inst.82:1107-1112 (1990)). At the time at which drugs were added assays wereperformed on extra reference plates to determine the cell populationdensity at time 0 (T₀). After 96 h incubation at 37 ° C. in a humidifiedatmosphere containing 5% CO₂, the assays were performed on test (T) andcontrol (C) plates. The absorbance of the wells was determined by aMicroplate Reader at 540 nm for CelITiter96™ and 490 for SRB. Cellularresponses were calculated from the data as described previously:100×[(T−T₀)/(C−T₀)] for T>T₀ and 100×[(T−T₀)/T₀] for T<T₀. (Monks etal., J. Natl. Cancer Inst. 83:757-766 (1991)). Results are presented inFIGS. 1-4, and as IC₅₀ and LC₅₀ values in Table 1.

Flow Cytometry Analysis. A suspension of 0.5×10⁶ cells in 8 ml of mediumwere placed in 25 cm² T flask and allowed to attach for 24 h. The cellswere then exposed for 6 h to 100 nM of drug . After removal of drug thecells were washed with PBS. Fresh medium (8 ml) was added and incubationat 37° C. in 5% CO₂ in complete humidity was continued for an additionalfive days. At appropriate intervals, treated and control cells werereleased from flasks by incubation with trypsin (0.05 mg/ml)/EDTA (0.02mg/ml) for 5 min at 37° C., collected in ice-cold PBS, combined with theremoved medium that might contain floating cells, and centrifuged at 4°C. Cell pellets were re-suspended in PBS containing 1% fetal bovineserum. The cells were fixed and stained for fluorescence-activated cellsorting according to standard procedures (Crissman et al., Cytometry3:84-90(1992)). Fluorescence histograms were obtained on a CoulterEPICS753 Cell Sorter using an argon laser and mean peaks were analyzed.

TABLE 1 Antitumor Activity in Cultured Cells Tumor cell lines CompoundIC₅₀ LC₅₀ IC₅₀ LC₅₀ IC₅₀ LC₅₀ HCT116 HT29 A549 Example 1 8 220 25 400 25500 Example 2 0.5 32 0.9 65 0.6 35 Example 4 0.5 35 1.5 35 0.6 35Example 6 2.2 80 8 750 2.5 100 Example 8 1.8 230 3.5 >1000 2 150Mitonafide 250 >1000 300 >1000 65 800 MELSK2 HL60 MCF7 Example 1150 >1000 120 500 50 >1000 Example 2 4 60 3.5 33 3.2 >1000 Example 4 470 3.5 33 3.5 >1000 Example 6 15 750 5 450 32 >1000 Example 8 20 >1000 4450 46 >1000 Mitonafide 150 700 45 400 245 >1000 IC50 - drugconcentration in nM which causes 50% cell growth inhibition LC50 - drugconcentration in nM which causes 50% cell death

Inhibition of DNA synthesis. The effect of test compounds on DNAsynthesis was examined by bromodeoxyuridine (BrdU) incorporation using aBrdU Cell Proliferation Assay (Oncogene Research Products, Cambridge,Mass.). In this assay 2,500 cells/well were allowed to attach for 24 h,treated with various concentrations of (hug for 24 h, and then incubatedwith BrdU for 24 h. The level of incorporated BrdU was measuredimmunochemically according to the manufacturer's protocol.

Viability assay. Cell death was additionally confirmed by the LIVE/DEAD™Viability/Cytotoxicity Kit (Molecular Probes, Eugene, Oreg.) appliedaccording to the manufacturer's Fluorescence Microscopy Protocolprovided with the kit.

3. Results

Compounds were examined in the NCI 60 human tumor cell line panel(Grever et al., Seminars in Oncology 19:622-638 (1992). In general, thecompounds were extremely active. For example, the compound of Example 2inhibited the growth of all of the tumor cell lines with a medianactivity in the nanomolar range. The activity of the compounds wasexamined in greater detail utilizing the MTT cell proliferation assay.This assay indirectly measures the number of living cells by measuringthe activity of the mitochondrial dehydrogenase. Briefly, tumor cellcultures, contained in a 96-well plate, are incubated with varyingconcentrations of the test chemical for various times. At the end of thetest period the wells are treated with the MTT dye solution andincubated for 4 hours. Cells which are alive convert the yellowtetrazolium dye into the blue, insoluble formazan product. Thisprecipitate is solubilized, and the absorption at 570 nm is read in anELISA reader.

FIG. 2 shows the activity of the compounds against two colon tumor celllines (HCT116 and HT29), the non-small cell lung cancer line A549, andthe melanoma line MelSK2, at 100 nM drug concentration. The cells wereincubated for 96 hours with the drug. It is clear from the Figure thatthe melanoma line was less sensitive in general than the other cancers,although the compounds of the invention did produce significant growtharrest. Note that mitonafide, a known agent that contains thenitronaphthalimide moiety, was not very active at this concentration. Atthe 100 nM concentration the compounds of Examples 2 and 4 exhibitedoutstanding cytotoxic activity against the three adenocarcinomas.Further in vitro dose-response studies were conducted with the colonline HCT116. FIG. 3 shows the resulting data in graphical form. Atconcentrations of 1 nM, the compounds of Examples 2 and 4 cause almostcomplete growth inhibition. Mitonafide did not show this effect until aconcentration of 1 μM was reached. Evidence of induced cytotoxicity withthe compounds was seen at 10 nM and, as shown in FIG. 3, substantialkilling was observed at 100 nM. These data suggest that the compounds ofthe invention have potent anti-tumor activity against tumors which arenormally difficult to treat.

To demonstrate that these data were not dependant on the assay systemused, the cytotoxic activity of Example 2 was examined against the fourtumor types using the sulforhodamine B (SRB) assay (Skehan et al., J.Natl. Cancer Inst. 82:1107-1112 (1990)). FIG. 4 shows the results ofthis assay, which essentially mirror the results obtained with the MTTend-point.

The compound of Example 2 is also potently cytotoxic against extremelyrefractory pancreatic cancer cell lines. FIG. 5 presents the results ofan SRB-based assay on the toxicity of Example 2 against the four celllines mentioned above, and against two pancreatic cell lines, 6.03 and10.05 (obtained from Dr. Elizabeth Jaffe, Johns Hopkins University,Baltimore Md.). The compounds were exposed to the agent for 120 hrs. Itis clear that both of these lines are sensitive to the compound. Themore resistant line 6.03, becomes growth arrested at 100 nMconcentration; the more sensitive line 10.05 suffers substantial celldeath at 1 μM.

In order to determine the mode of cell death induced by the compounds ofthe invention, the effect of the compound of Example 2 on the cell cyclewas studied, utilizing Fluorescence Activated Cell Sorting SACS)analysis. HCT 116 colon cancer cell were treated with the compound ofExample 2 for 6 hrs at a concentration of 100 nM, and then allowed togrow in culture for varying times. As early as 24 hrs, the cells becamegrowth arrested at G₂-M and some sub-G₁ cells began to appear. Thisfraction, which is associated with apoptotic death, increased steadilywith time, and dominated the distribution at 96 hrs. Untreated cellsbecome growth arrested at G₁ at 96 hrs as they reach confluence.

Similar cell cycle experiments were carried out on the 10.05 pancreaticcancer line. Dramatic differences were apparent at 48 hrs, and it wasclear that substantial apoptosis was occurring at 144 hrs, as evidencedby the growth of the sub-G₁ fraction. Similar observations were madewith the slower growing 6.03 pancreatic cancer cells, which also showedstrong evidence of apoptosis.

The FACS analysis evidence of apoptosis was substantiated bymorphological examination of the treated 10.05 pancreatic cancer cells.The treated cells showed evidence of chromatin fragmentation, which wasabsent in the untreated cells. Similar results were obtained with the6.03 cells.

The results strongly indicate that the compounds of the invention arepotent, selective new cytotoxic agents which are active against tumorsthat are normally not sensitive to chemotherapeutic agents, and that theunsymmetrical bifunctional intercalators of this invention offer newpossibilities for the treatment of cancer.

While the examples presented above describe a number of embodiments ofthis invention, it is apparent to those skilled in the relevant artsthat the compounds, compositions, and methods of this invention can bealtered to provide alternative embodiments, and equivalent compositionsand methods, which nonetheless remain within the scope of the invention.Therefore, it will be appreciated that the present invention is notlimited in scope by the specific embodiments described above, which aremerely illustrations of individual aspects of the invention. Inparticular, modifications which are obvious to those of ordinary skillin the art are intended to be within the spirit and effective scope ofthe following claims.

We claim:
 1. A compound of structure 1:

wherein A is (CH₂)_(m) or (CHR)_(m); B is NR′, NR′(CH₂)_(n)NR″,hexahydropyrimidine-1,3-diyl, piperazine-1,4diyl,4-aminopiperidine-1,4N-diyl, or 1,4-diazacycloheptane-1,4-diyl; D is(CH₂)_(p) or (CHR)_(p); Y¹, Y², Y³ and Y⁴ are independently R, COR,CO₂R, CONRR′, SR, SOR, SO₂R, SO₂CF₃, SO₂NRR′, OR, OCF₃, OCOR, OCONRR′,NO₂, NRR′, CN, Ph, CF₃, NRCOR′, NRCONR′R″, NRC(NR′)NR″R′″, NRCOCF₃,NRSO₂R′, NRSO₂CF₃, or halogen; R is H, CF₃, lower alkyl, amino-loweralkyl, or hydroxy-lower alkyl; R′ and R″ are independently R, C(O)R, orSO₂R; and m, n, and p are independently 2-6.
 2. A compound according toclaim 1, wherein B is piperazine-1,4-diyl.
 3. A compound according toclaim 1, wherein Y¹, Y², Y³ and Y⁴ are independently chosen from thegroup consisting of H, F, Cl, OR, NH₂, NO₂, SO₂CF₃, CN, and CF₃.
 4. Acompound according to claim 2, wherein Y¹, Y², Y³ and Y⁴ areindependently chosen from the group consisting of H, F, Cl, OR, NH₂,NO₂, SO₂CF₃, CN, and CF₃.
 5. A compound according to claim 1, selectedfrom the group consisting of: 2-{3-{methyl[3-(6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]amino}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{methyl[3-(8-fluoro-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]-amino}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{methyl[3-(8-hydroxy-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]-amino}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{methyl[3-(8-trifluoromethyl-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]amino}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{methyl[3-(6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]amino}propy}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione:2-{3-{methyl[3-(8-fluoro-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]-amino}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{methyl[3-(8-hydroxy-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]-amino}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione;and2-{3-{methyl[3-(8-trifluoromethyl-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]amino}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione.6. A compound according to claim 2, selected from the group consistingof:2-{3-{4-[3-(6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{4-[3-(8-fluoro-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{4-[3-(8-hydroxy-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]-piperazin-1-yl}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{4-[3-(8-trifluoromethyl-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{4-[3-(6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{4-[3-(8-fluoro-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione;2-{3-{4-[3-(8-hydroxy-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione;and2-{3-{4-[3-(8-trifluoromethyl-6-oxo-6H-imidazo[4,5,1-de]acridin-5-yl)aminopropyl]piperazin-1-yl}propyl}-5-amino-1H-benz[de]isoquinoline-1,3(2H)-dione.7. A method of preparing a compound according to claim 1, comprising thestep of contacting a compound of structure

with a compound of structure

in a suitable inert solvent.
 8. A method of preparing a compoundaccording to claim 1, comprising the step of contacting a compound ofstructure

with a compound of structure

in a suitable inert solvent.
 9. A method of preparing a compoundaccording to claim 1, comprising the step of contacting a compound ofstructure

with a compound of structure

wherein Z is F, Cl, or another leaving group, in a suitable inertsolvent.
 10. A method of inhibiting the proliferation of mammaliancells, comprising exposing said cells to an effective amount of acompound of claim
 1. 11. A method of inhibiting the proliferation ofmammalian cells, comprising exposing said cells to an effective amountof a compound of claim
 2. 12. A method of inhibiting growth of a tumor,comprising exposing said tumor to an effective amount of a compound ofclaim
 1. 13. A method of inhibiting growth of a tumor, comprisingexposing said tumor to an effective amount of a compound of claim
 2. 14.The method of claim 12, wherein the tumor is an adenocarcinoma.
 15. Themethod of claim 13, wherein the tumor is an adenocarcinoma.
 16. A methodof treating a mammal suffering from cancer, comprising administering tosaid mammal an effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof.
 17. A method of treating amammal suffering from cancer, comprising administering to said mammal aneffective amount of a compound of claim 2, or a pharmaceuticallyacceptable salt thereof.
 18. A pharmaceutical composition comprising aneffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, further comprising one or more pharmaceuticallyacceptable additives selected from the group consisting of carriers,preservatives, flavorants, excipients, fillers, wetting agents, binders,disintegrants, and buffers.
 19. A pharmaceutical composition comprisingan effective amount of a compound of claim 2, or a pharmaceuticallyacceptable salt thereof, further comprising one or more pharmaceuticallyacceptable additives selected from the group consisting of carriers,preservatives, flavorants, excipients, fillers, wetting agents, binders,disintegrants, and buffers.
 20. A pharmaceutical composition comprisingan effective amount of a compound of claim 3, or a pharmaceuticallyacceptable salt thereof, further comprising one or more pharmaceuticallyacceptable additives selected from the group consisting of carriers,preservatives, flavorants, excipients, fillers, wetting agents, binders,disintegrants, and buffers.